The persistence of the latent reservoir in HIV-infected patients is a major barrier to the eradication of this disease. Although combination anti-retroviral therapy (cART) is highly effective at suppressing viral production from the latent reservoir, cART withdrawal leads to a rapid viral rebound driven by the reactivation of viral transcription from the latent pool. In this project, we will develop a multi-targeted therapy that we expect will significantly reduce and potentially eliminate the ability of HIV-1 to infect target cells in the absence of cART. This combination therapeutic will target different key aspects of HIV infection, each of which contributes to sustained viremia in the absence of cART. These components include viral particles, virus producing cells, the CCR5 co-receptor expressed on target cells, and the integrated provirus. To clear HIV and HIV-producing cells that emerge during viral rebound, we have engineered a panel of anti-HIV monoclonal antibodies. These antibodies bind to various epitopes on HIV- 1 and have been modified to express functionally enhanced Fc domains. These altered Fc domains promote Fc receptor functions on myeloid cells such as antibody dependent cellular cytotoxicity (ADCC), phagocytosis, and complement recruitment. We have developed CRISPR/Cas constructs that target and cleave the CCR5 gene. CCR5 gene cleavage in the CD34+ hematopoietic stem cell (HSC) population will lead to the differentiation and proliferation of CCR5-negative immune progeny that are resistant to infection with R5-tropic HIV-1. Moreover, CCR5 gene cleavage in differentiated leukocytes will also result in HIV-1 resistance. We have developed CRISPR/Cas gene editing constructs to cleave the integrated proviral sequence, so that infected cells transduced with these genes are unable to transcribe intact viral genomes. To deliver these CRISPR/Cas transgenes, we have developed lentiviral (LV) vectors that we will pseudotype with different viral envelope proteins and anti-receptor antibodies to direct LV to specific target cells. CRISPR genes delivered by LV are stably integrated into the target cell genome so that the transduced cells and their progeny are permanently protected from HIV infection. Typically, each of these approaches would be proposed independently, and efficacy measured against the absence of treatment. However, a key innovation of this application is to combine these approaches into one therapeutic regimen. In the first 3 aims, we will develop and select the most efficacious reagents for each of these approaches using a combination of in vitro cellular assays and in vivo assessments in the HIV-infected humanized mouse (hu-mouse). In Aim 4, we will combine the therapeutics and test them in the HIV-infected hu-mouse model, and determine their ability to suppress and delay viral production following interruption of cART treatment. We expect that these studies will not only develop clinic-ready reagents that will have undergone rigorous testing and validation in a pre-clinical animal model, but we will also demonstrate that combination targeting, like combination anti-retroviral therapy, is more effective than a single therapeutic in reducing the viral burden in HIV patients and in developing an HIV-resistant immune system. This work brings together the combined expertise of Drs. Susan Eszterhas and George O'Toole in the design of CRISPR gene targeting constructs, Dr. Bryan Luikart in the development of LV vectors for primary cell targeting in vivo, Dr. Margaret Ackerman in the optimization of antibodies for therapeutic development, and Dr. Dorothy Wallace in mathematical modeling of human diseases.